There exists a motivation to decrease the overall cost and size of flow cytometry equipment, particularly in laboratory and industrial applications, such as in sperm sorting. Currently, sperm sorting and other flow cytometry applications operating at ultraviolet wavelengths require expensive, bulky, energy high consumption excitation sources. In sperm sorting and other UV applications a significant demand is placed on the laser for obtaining sufficient laser power, at an appropriate wavelength for optimum excitation with a uniform spatial beam profile, and with sufficiently low noise to provide the necessary fidelity required to resolve minor differences between various particles. Recent developments in solid state and diode lasers have held much promise for replacing commercial lasers with smaller, more efficient, lower cost lasers, however the combined power, wavelength, spatial uniformity, and cost requirements of sperm sorting flow cytometer systems have not yet been met by these lasers.
Nichia (a Japanese Corporation) released the first commercially available ‘high power’ (i.e. >20 mW) UV laser diode as engineering samples. However, the specifications of this system, at glance, appear unsuitable for certain applications, such as sperm sorting. The 370 nm to 380 nm, 200 mW system does not appear capable of meeting the power and profile requirements for making high resolution DNA content measurements. Such lasers appear unsuitable for sorting sperm because of the presence of multiple simultaneous lasing modes that result in a highly irregular, or variable, spatial intensity pattern across the laser beam profile (perpendicular to the axis of propagation). These unfavorable attributes are expected to make the released UV diode laser unsuitable for application to sperm DNA content measurements. Further, while the 200 mW power output specified may suggest that such an excitation source might be a drop-in replacement for a 150 mW pulsed 80 MHz NdYAG laser operating at 355 nm, the excitation efficiency of the primary fluorescent dye (Hoechst 33342) used for sperm sorting drops from near 100% (at 355 nm) to approximately only 50% over the 370 to 380 nm range. Thus, in order to match the energy delivered by the NdYAG laser to achieve similar excitation power and fluorescence response (to obtain a good X-Y ‘split’), one would require a 50% increase in the 200 mW provided in this unit. Further restricting the potential for using such a system, once assembled into an integrated laser module, it is difficult to maintain a full 200 mW with coupling losses, and if laser diode lifetime is a concern, it is recommended that lasers be operated at input currents that produce even lower powers to avoid premature component failure.